Type 2 diabetes mellitus (T2DM) and coronary artery disease (CAD) are leading causes of morbidity and mortality worldwide. Development of new and more effective approaches to prevention and treatment requires improved understanding of disease mechanisms. Genetic mapping in humans offers an approach to identify genes and DNA variants underlying the inherited contribution to disease susceptibility, unbiased by prior assumptions about the pathophysiological processes responsible. Next-generation sequencing technologies make it possible for the first time to catalog mutations observed in patients and in healthy controls. While human genetics has the potential to dramatically expand our knowledge of biological and disease mechanisms, progress is constrained by two central challenges: (a) determining which DNA changes are functional, and which are benign, and (b) developing cellular assays with which to interrogate the functions of the genes and variants thereby identified. Specifically, the field requires methods to functionally screen large number of mutations in high throughput, using assays that faithfully represent the human cell types of interest. This proposal is built on a foundation of human genetics, genome engineering, and stem cell biology, and is focused on the major metabolic diseases of T2DM, dyslipidemia, and CAD. The approach leverages three recent advances: (a) ongoing next-generation sequencing studies identifying candidate disease mutations, (b) development of methodologies to rapidly alter genes of interest in human pluripotent stem cells (hPSCs) using engineered TAL effector nucleases (TALENs), and (c) protocols to differentiate hPSCs into cell populations with characteristics of hepatocytes, adipocytes, and pancreatic beta cells. Our proposal combines two central innovations. First, we propose to develop two novel approaches to engineer the genomes of hPSCs, rapidly and efficiently knocking out the function of candidate genes, and introducing specific mutations observed in patients. This will generate isogenic human stem cells that differ only at a single mutation of interest. Second, we will develop and improve protocols to differentiate hPSCs into physiologically mature hepatocytes, adipocytes, and beta cells. By engineering stem cells to carry specific mutations, and by differentiating these engineered stem cells into physiologically relevant human metabolic cell types, we will make it possible to study the impact of large numbers of gene variants on human cell biology and function. By relating the functions of gene mutations and cell biological processes with the phenotypes of human patients, we will provide pathophysiological insights and practical in vitro assays to guide development of therapeutics for these challenging diseases. PUBLIC HEALTH RELEVANCE: Next-generation genome sequencing has made it possible to discover gene mutations in patients with type 2 diabetes and heart attack, two of the leading causes of death and disability worldwide. To determine the functional relevance of these gene mutations, we have developed an approach to insert specific mutations into human stem cells, and to grow these engineered cells into tissues that are involved in disease such as liver, fat, and insulin-producing cells of the pancreas. By relating the functional effects of the mutations to the disease characteristics of the patients who carry them, we will provide new insights into the molecular and cellular causes of type 2 diabetes and heart attack, and provide new and more relevant laboratory assays with which to develop new therapies.